STRUCTURAL INSTABILITY OF A CONSTITUTIVELY ACTIVE G-PROTEIN-COUPLED RECEPTOR - AGONIST-INDEPENDENT ACTIVATION DUE TO CONFORMATIONAL FLEXIBILITY

Mutations in several domains can lead to agonist-independent, constitu tive activation of G protein-coupled receptors. However, the nature of the structural and molecular changes that constitutively turn on a G protein-coupled receptor remains unknown. Here we show evidence that a constitutively activated mutant of the beta(2) adrenergic receptor (C AM) is characterized by structural instability and an exaggerated conf ormational response to ligand binding. The structural instability of C AM could be demonstrated by a 4-fold increase in the rate of denaturat ion of purified receptor at 37 degrees C as compared with the wild typ e receptor. Spectroscopic analysis of purified CAM labeled with the co nformationally sensitive and cysteine-reactive fluorophore, 7-nitroben z-2-oxa-1,3-diazol-4-yl)ethylenediamine, further indicated that both a gonist and antagonist elicit more profound structural changes in CAM t han in the wild type protein. We propose that the mutation that confer s constitutive activity to the beta(2) adrenergic receptor removes som e stabilizing conformational constraints, allowing CAM to more readily undergo transitions between the inactive and the active states and ma king the receptor more susceptible to denaturation.